Biomarkers for tuberculosis: the case forlipoarabinomannan

Margarida Correia-Neves1,2,3, Gabrielle Fröberg 3,4, Liudmyla Korshun5,Sofia Viegas6, Paula Vaz6,7, Nehaben Ramanlal7, Judith Bruchfeld3,4,Beston Hamasur5,8, Patrick Brennan9 and Gunilla Källenius 3

Affiliations: 1Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho,Braga, Portugal. 2ICVS/3B’s, PT Government Associate Laboratory, Braga/Guimarães, Portugal. 3Division ofInfectious Diseases, Dept of Medicine Solna, Karolinska Institutet, Stockholm, Sweden. 4Dept of InfectiousDiseases, Karolinska University Hospital, Stockholm, Sweden. 5Biopromic AB, Solna, Sweden. 6InstitutoNacional de Saúde, Ministério da Saúde, Maputo, Mozambique. 7Fundação Ariel Glaser Contra o SIDAPediátrico, Maputo, Mozambique. 8Dept of Microbiology, Tumor and Cell Biology, Karolinska Institutet,Stockholm, Sweden. 9Dept of Microbiology, Immunology and Pathology, Colorado State University, FortCollins, CO, USA.

Correspondence: Gunilla Källenius, Division of Infectious Diseases, Dept of Medicine Solna, KarolinskaInstitutet, Stockholm 17176, Sweden. E-mail: gunilla.kallenius@ki.se

ABSTRACT Tuberculosis (TB) is considered the most onerous of infectious diseases according to recentreports from the World Health Organization. Available tests for TB diagnosis present severe limitations,and a reliable point-of-care (POC) diagnostic test does not exist. Neither is there a test to discern betweenthe different stages of TB, and in particular to predict which patients with Mycobacterium tuberculosisinfection and no clinical signs are more at risk of advancing to overt disease. We here review theusefulness of mycobacterial lipoarabinomannan (LAM) as a diagnostic marker for active and latent TBand, also, aspects of the immune response to LAM relevant to such tests. There is a high potential forurinary LAM-based POC tests for the diagnosis of active TB. Some technical challenges to optimisedsensitivity of the test will be detailed. A method to quantify LAM in urine or serum should be furtherexplored as a test of treatment effect. Recent data on the immune response to LAM suggest that markersfor host response to LAM should be investigated for a prognostic test to recognise individuals at thegreatest risk of disease activation.

@ERSpublicationsThere is a high potential for a urinary LAM-based point-of-care test to diagnose TB. Markersfor host response to LAM should be explored to identify those at highest risk of developingactive TB. http://ow.ly/FyCs30n4uFE

Cite this article as: Correia-Neves M, Fröberg G, Korshun L, et al. Biomarkers for tuberculosis: thecase for lipoarabinomannan. ERJ Open Res 2019; 5: 00115-2018 [https://doi.org/10.1183/23120541.00115-2018].

Copyright ©ERS 2019. This article is open access and distributed under the terms of the Creative Commons AttributionNon-Commercial Licence 4.0.

Received: July 20 2018 | Accepted after revision: Nov 05 2018

https://doi.org/10.1183/23120541.00115-2018 ERJ Open Res 2019; 5: 00115-2018

REVIEWTUBERCULOSIS

Tuberculosis: a global threat to human healthIt is estimated that about 23% of the global population is infected with Mycobacterium tuberculosis [1] andtuberculosis (TB) accounts for 1.4 million deaths annually and for one-fifth of adult deaths in poor/low-income countries. Each patient with active pulmonary TB, if left untreated, is estimated to infect10–15 other individuals per year [2]. Thus, interrupting disease transmission is of major importance andrequires early detection, in combination with adequate treatment.

Host immune response in TBDepending on the immune response of the host, upon exposure to M. tuberculosis, individuals mighteliminate the bacteria, progress to active TB or develop a persistent infection in the absence of clinicalmanifestation, referred to as latent TB. The initial response is due to the interaction of the unique lipids/glycolipids/carbohydrates/peptidoglycans of the M. tuberculosis cell envelope, with cells of the innateimmune system such as macrophages and dendritic cells [3]. The manner in which macrophages anddendritic cells activate or suppress distinct microbicidal mechanisms, the pattern of cytokine beingproduce and secreted, and how antigens interact with the major histocompatibility complex dictates theprofile of the acquired immune response. The elicited acquired immune response mediated by T-cellsplays a very important part in M. tuberculosis infection control [4]. However, the precise M. tuberculosisantigens and detailed profile of the host immune response necessary for effective acquired immunity toM. tuberculosis have yet to be determined. Most studies of the acquired immune response focus on therole played by antigenic proteins/peptides and very little address the mycobacterial antigens of a lipoglycannature. However, lipoglycan antigens may have been undervalued and might in fact play a crucial part inthe overall immune response to M. tuberculosis and as such be of extreme value for TB diagnosis, a subjectdiscussed further ahead.

While the importance of T-cell immunity is long established, the role of humoral immunity has beenconsidered controversial. There is however increasing recent evidence supporting a role for antibodies andB-cells in the establishment of an effective immune response against M. tuberculosis infection [5–7].During active TB, antibody responses are prominent [8, 9], and antibody levels to particularM. tuberculosis protein antigens may increase before symptoms of active TB [8]. Although people withactive TB have been shown to produce antibodies with a low affinity to M. tuberculosis surface moleculesand with a low ratio of IgG/IgM [10], there is evidence suggesting that specific antibodies might preventM. tuberculosis dissemination. Antibodies in the mucosa may also potentially prevent infection via thisroute [11]. Elevated Ag85A-specific IgG titres have recently been identified as a correlate of a lower risk ofTB disease in the MVA85A vaccine trial [12], indicating a possible role for antibodies in M. tuberculosisprotective immunity. Here also, antibodies specific to mycobacterial glycolipids seem to play a relevantrole [13–15] as discussed in detail later.

The need for better TB diagnostic strategies and novel biomarkersPresently available markers/tests for TB diagnosis exhibit serious limitations, and none is a point-of-care(POC) diagnostic test. There is an intensive search for diagnostic biomarkers for TB [16–18], as well aspredictive markers for progression from latent to active TB [19]. It is increasingly evident that latent TBshould be viewed as part of a continuous spectrum, extending from sterilising immunity, to persistentnonprogressing infection and subclinical infection progressing to active disease [20, 21]. Available tests areunable to distinguish those patients with subclinical progressing infection from those with nonprogressinglatent infection [22–24]. Here we discuss the potential use of LAM in the diagnosis of active TB, inpredicting the outcome following M. tuberculosis infection and in response to treatment.

Recently the World Health Organization (WHO) specified the target product profile (TPP) of the mosturgently needed tests for TB [25]. With the objective of initiating treatment, the specificity should be 98%and for sensitivity the minimum requirements should be an overall sensitivity ⩾65%. Besides the need fora diagnostic test, WHO defined as one of the highest priorities a POC triage test, for systematic screening,able to distinguish individuals who need further confirmatory testing. The TPP for such a test was definedby WHO as overall specificity >70% (optimally >80%) and sensitivity >90% (optimally >95%). To achievethese goals the sensitivity of present tests should be increased, while retaining the high specificity. Inaddition, in line with the WHO goal of TB elimination by 2050, there is a great need for a test thatidentifies patients with latent TB who are on the rise of developing active disease.

Diagnostic laboratory methods for diagnosis of active TB are based on detection of evidence ofM. tuberculosis by acid fast staining or direct culturing of bacilli in sputum specimens. Microscopy lackssensitivity, while mycobacterial culturing, the ultimate diagnostic test for TB, is a prolonged exercise thatmay require up to 6–8 weeks, or even more, to provide results. The GeneXpert MTB/RIF (Cepheid Inc.,

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Sunnyvale, CA, USA), and more recently Xpert Ultra, a superior new generation version of GeneXpert,have become important additions to standard liquid culture.

Diagnosis of the latent forms of TB relies on the tuberculin skin test (TST) or the interferon-γ releaseassays (IGRAs). The TST uses purified tuberculin derivative (PPD), which is nonspecific forM. tuberculosis. The interpretation of a TST needs to take into consideration false positive results inindividuals exposed to ubiquitous non-TB mycobacteria and/or previously vaccinated with BCG. Thecommercial IGRAs (Quantiferon Gold-IT and more recently Quantiferon Plus (QIAGEN, Venlo, theNetherlands) as well as T-spot.TB (Oxford Immunotec, Marlborough, MA, USA) useM. tuberculosis-specific antigens (ESAT-6 and CFP-10) and include test tubes to control for anergy andbackground reactivity. However, neither the TST nor IGRAs can help identifying which of the patientswith latent TB are on the verge of progression to active TB, those in immunological control ofM. tuberculosis nor the ones that exhibit just immunological memory to a previous M. tuberculosisinfection. Thus, although these tests are useful in patient management they have no predictive value forprogression to active TB [22–24, 26, 27].

Ideal diagnostic tests would distinguish between those with actual clinical TB; those with the potentialreactivation/latent form; and those who have been cured. For the usage of the test in all different settings,less intrusive sources of potential biomarkers such as human urine, would be ideal.

An example of a new strategy that could be applied for both latent and active TB lies on thedetermination of particular circulating B lymphocytes in the peripheral blood [28]. It has been shownthat B lymphocytes may discriminate individuals with latent and active TB, namely the ratio ofplasmablast to memory lymphocytes. Interestingly LU et al. [29] reported recently that latent TB isassociated with particular M. tuberculosis-specific antibody Fc functional profiles, and distinct antibodyglycosylation patterns. Antibodies from patients with clinical evidence of TB when compared with thoseconforming to the definition of latent TB, were found to drive phagolysosomal maturation,inflammasome activation, and macrophage killing of intracellular M. tuberculosis [29]. These resultsstrongly suggest a role for Fc-mediated antibody effector functions for an effective immune response toM. tuberculosis [29].

Blood RNA signatures prospectively identified people at risk of developing active TB with a sensitivity of53.7% (42.6–64.3) and a specificity of 82.8% (76.7–86.0) [30]. Interestingly, the same signature was laterfound in four different African countries [31]. This finding strongly validates the potential usefulness ofthe blood RNA signatures as an important tool to identify individuals at higher risk of progression toactive disease.

A different strategy that holds promise is untargeted metabolomic approaches. However, the majority ofmetabolites in human body fluids that could accurately identify patients early in infection have lackedstructural definition [32]; an intriguing exception is the identification of N1-acetylisoputreanine anapparent novel polyamine metabolite in human urine that may have potential as a host-derived biomarkerof M. tuberculosis infection [33].

In a proteomic approach to discover biomarkers for pulmonary TB in urine samples, 10 mycobacterialproteins were identified as unique biomarkers of the active from of TB whereas a different set of 6 proteinswere characteristic of latent TB; partial identifications of these proteins were achieved, notably, isoforms of thePE-PGRS protein family were prominent in both the proteins from patients with active and latent TB [34].

LAM as a biomarker for TB diagnosisLAM, a major lipoglycan (a multiglycosylated extension of the phosphatidylinositol mannoside (PIM)glycolipids) of the mycobacterial cell envelope has been shown to be present in the urine ofM. tuberculosis-infected individuals.

LAM is a major constituent of the M. tuberculosis cell wall/envelope and represents up to 15% of thebacterial mass. It is composed of a mannan “core” (an extension of the PIM structure) which is decoratedby a single branched arabinan chain. In addition, in the case of M. tuberculosis the majority of theterminal arabinose unity of the arabinan chain have attached to them short 2–4 mannose containingoligosaccharides called “mannose capping motifs”; this form of LAM is known as ManLAM [35–37](figure 1). LAM exhibits important and distinct immunomodulatory properties [40, 41]. It is considered avirulence factor associated with the pathogenesis of M. tuberculosis infection. During mycobacterialinfections, LAM is present in body fluids representing a potential biomarker to identify infectedindividuals. In addition, the immune response to LAM might also serve as a diagnostic tool. Theusefulness of LAM as a diagnostic marker will be discussed in the following sections.

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Detection of LAM in body fluids as a method for TB diagnosisTests that detect M. tuberculosis antigens in clinical specimens would potentially offer several advantagesin terms of a specific biomarker of TB [42]. Tests for urinary LAM have generated great enthusiasm andnumerous publications. However, several difficulties have been encountered over the last two decades andthe consensus is that the present test(s) are not sensitive enough to diagnose latent TB nor active TB inHIV-negative patients [43–46]. However, recent results strongly suggest that this is a path that deservesfurther effort. A brief historical perspective will be presented followed by the results obtained with twocommercially available tests and the efforts presently ongoing to render the use of LAM detection in urine,and potentially other body fluids, a useful diagnostic test for TB.

The first reports on the possible use of LAM in urine as a diagnostic test were published in 2001 by ourteam. We showed that LAM is present in urine from individuals with active TB [47–49] as well as miceinjected with sonicated M. tuberculosis [47]. Although we clearly showed that LAM is present in urine ofpatients with active TB, the method used at that time required a laborious concentration step. Thesensitivity achieved by us, as well as by several subsequent groups, did not reach the sensitivity requiredfor a diagnostic test for TB.

Two tests were developed by companies and commercialised but obvious improvements are needed torender this approach of general use for the patients in need. A sandwich ELISA, employing polyclonalantibody preparations, was first marketed by Chemogen Inc. (South Portland, ME, USA) as the“Mtb ELISA” test and was sold as “Clearview TB ELISA” first by Inverness Medical Innovations andlater by Alere Inc (Waltham, MA, USA). A set of clinical studies [50–55] using these commercial testswere performed, resulting in a pooled estimated sensitivity of 14% for patients with active TB and noHIV infection and 51% for patients co-infected with HIV (table 1) [56]. Subsequently a lateral flow test,the Determine TB-LAM Ag (Determine TB-LAM; Alere Inc.), using the same polyclonal antibody

FIGURE 1 Schematic rendition ofthe structure of Mycobacterium spp.LAM. Only the major commonstructural features are shown (seevarious works for more structuraldetail [38, 39]). LAM: lipoarabino-mannan. LAM

D-arabinofuranose

D-mannopyranose

D-myoinositol

Succinyl

Palmitic acid

Tuberculostearic acid

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preparations as in the Clearview ELISA, was produced as a POC test. Although easier to perform, thesensitivity of that test remained disappointingly low, as demonstrated in numerous clinical studies. In asystematic review [57] of 12 studies, 6 evaluated the Determine TB-LAM test to identify patients withactive TB and 6 others evaluated the test for TB screening in HIV-positive adults. It was found thatDetermine TB-LAM has low sensitivity for both diagnosis and screening. The combination of DetermineTB-LAM with sputum microscopy revealed an increased sensitivity for active TB when compared witheither test alone but accompanied by a decreased specificity. Determine TB-LAM was shown to potentiallyimprove the accuracy of active TB diagnosis only for seriously ill patients with HIV-co-infection and lowCD4+ T-cell counts [57].

These tests were most sensitive for patients with profound immunosuppression revealing an inversecorrelation between CD4+ T-cell counts and urine LAM sensitivity [57–59] (figure 2). Pooled sensitivity ofDetermine TB-LAM was 56% (41–70%) and specificity 90% (81–95%) in participants with CD4+ T-cellcounts below or equal to 100 cells·µL−1 [57] with a urinary LAM concentration in the range of ng per mL [60].LAM positivity has been associated with poor prognosis [61] and with increased mortality [44, 62–65] andhas been reported as an independent predictor of mortality [63]. In addition, the usage of bedside LAMtesting as a guide for initiating anti-TB treatment in HIV-infected hospitalised inpatients has beenassociated with decreased mortality [66]. After a long evaluation process, WHO recommended in 2016that the Determine TB-LAM assay might be utilised for TB diagnosis in a small subgroup of patients (i.e.in HIV-infected hospitalised adults with symptoms and signs of TB who either are seriously ill and/orhave CD4+ T-cell counts below or equal to 100 cells·μL−1). Recently, in a randomised controlled trial of2600 HIV-infected hospital inpatients with TB, the addition of urine LAM-based screening using the AlereTB-LAM test, a significant mortality reduction was observed in three subgroups of patients: those with lowCD4+ T-cell counts, severe anaemia or clinically suspected TB [67].

The high, and thus detectable, LAM concentration in urine of patients with advanced immunodeficiencyhas been postulated to depend on a high mycobacterial burden [68] and systemic dissemination ofM. tuberculosis. It seems reasonable that patients with a more confined TB would excrete less LAM.Another interpretation could be that the renal tract is affected in advanced HIV infection with

TABLE 1 Performance of commercial urinary lipoarabinomannan (LAM) tests

Sensitivity % Specificity %

TB-LAM (Clearview) ELISA [56]HIV-positive patients 51 (32–69) 94 (79–98)HIV-negative patients 14 (7–24) 97 (90–99)

Determine (Alere) TB-LAM test [57]HIV-positive patients 45 (29–63) 92 (80–97)HIV-negative patients ND ND

Data are presented as median (range). TB: tuberculosis; ND: not determined.

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FIGURE 2 Correlation between CD4+ T-cell counts and lipoarabinomannan (LAM) detection in urine.Compilation of results from a meta-analysis of 12 different studies of tuberculosis (TB)-LAM for TB diagnosisand screening. Plots of a) sensitivity and b) specificity of TB-LAM stratified by CD4+ T-cell counts. Circlesrepresents the pooled estimates (median), with bars representing 95% credible intervals. Reproduced from[57] with permission from the publisher.

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accompanying mycobacteriuria [69–71]. More studies are required to reach consensus on this topic. Thefinding of LAM in urine prior to the initiation of antiretroviral therapy has also been reported to beassociated with immune reconstitution inflammatory syndrome (IRIS) [60, 72], indicating that LAMdetection in urine may predict the development of TB IRIS. The association of urinary LAM and IRISsupports the idea that high urinary LAM concentration reflects a disseminated disease with high bacterialload. Considering that LAM in urine might, at least in part, reflect the bacterial load in the body,measurement of LAM concentration has been suggested as a tool to monitor the effectiveness of anti-TBtreatment (figure 3) [46, 64, 69].

Patients with latent or incipient TB who are on the verge of developing active TB, hypothetically carrybacteria that are metabolically active and dividing. Accordingly, one expects these individuals to excretesmall amounts of LAM in urine, a hypothesis that needs to be tested with more clinical research. Ifconfirmed, a more sensitive urinary LAM assay could possibly become a test to identify individuals atincreased risk of progressing to active disease.

Detection of LAM in other body fluids has also been investigated. A few efforts have been made to detectLAM in sputum, without much success [58, 74], mainly depending on the viscosity and interfering matrixof the sputum. LAM was rarely detected in cerebrospinal fluids of patients with TB meningitis [75, 76] orin pleural effusions [77]. With respect to extrapulmonary TB, tests for LAM in urine were rarely positivein one study of meningitis cases [78].

Host immune responses to LAM as biomarkers for TBThe source of LAM used in in vitro tests is of utmost importance for the correct dissection of theimmune response to LAM. ManLAM derived from M. tuberculosis strain H37Rv and clinicalM. tuberculosis isolates elicit proinflammatory cytokine responses in human macrophages and dendriticcells [3, 79, 80], while ManLAM from the laboratory strain M. tuberculosis Erdman induces no or just aweak response composed of pro- and anti-inflammatory cytokines [81]. Contamination with, for example,PIMs during the preparation of ManLAM may be the cause of some of the reports showing a weakproinflammatory response for particular ManLAM preparations [79]. Most studies of the adaptiveimmune response against M. tuberculosis have focused on the response to antigenic proteins/peptides andvery little has been done on mycobacterial antigens of carbohydrate nature, in spite of the fact thatmycobacterial glycolipids/lipoglycans are the dominant structural entities of the mycobacterial cell walland have strong and distinct effects on the immune response. Only a few reports show the effect of LAMon the adaptive immune response. Liposomal delivery of LAM was shown to trigger M. tuberculosis-specificT-cell activation [82]; polycytotoxic T-cells from bronchiolar lavage were recently reported to be induced bymycobacterial glycolipids, in particular by LAM, which was associated with protection against M. tuberculosisinfection [83].

Identifying specific markers or cytokine/chemokines of cell activation induced by LAM is an interestingvenue to explore as an alternative to the present IGRA tests, in particular in the differentiation between

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FIGURE 3 Signal intensity of Mycobacterium tuberculosis lipoarabinomannan in urine of children withtuberculosis (TB) in a high TB/HIV setting by ELISA at different time points after initiation of anti-TB treatment.Optical density (OD) results for HIV-positive participants are shown in red solid lines, results for HIV-negativeparticipants as black dashed lines. The signal intensity became undetectable after 3 months of treatment insix participants. y-axis shows logarithmic scale for mean OD. Neg.: negative. Reproduced from [73].

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different stages of latent TB as well as a complement for active TB diagnosis. Thus, there is an intensesearch for alternatives to ESAT-6 and CFP-10 as antigens used in the IGRA tests [84], as well asalternative to interferon (IFN)-γ [85–87].

Anti-LAM antibodies as markers of TB diseaseAnti-LAM antibodies are induced both during TB infection [88, 89] and after BCG vaccination [89–91].These antibodies have been associated with M. tuberculosis opsonisation and restriction of intracellulargrowth [90, 91]. Passive transfer of anti-LAM monoclonal antibodies has been shown to confer protectionin mice [13, 14]. Based on these findings a number of commercial tests based on antibody detection weredeveloped [92], but none proved to be specific and sensitive enough. In a policy statement in 2011, WHOrecommended against the usage of these tests while underscoring the relevance of continued investigationof diagnostic strategies based on antibody detection [93]. The majority of these commercial tests weredirected at antibodies specific for protein antigens, except one, the Mycodot test (DynaGen Inc.,Cambridge, MA, USA), that detects antibodies specific for LAM. Studies with the Mycodot test inTanzania, Guinea Bissau and Japan revealed sensitivities and specificities from 16.0% to 67.5% and 84.0%to 97.5%, respectively [94]. Using capsular arabinomannan, presumably devoid of the phosphatidyl inositolanchor and its constituent fatty acids, but otherwise structurally identical, anti-arabinomannan antibodytitres were significantly higher for TB individuals that were HIV-negative than for the ones that were HIVco-infected in one study in Tanzania (p<0.01) [95]. In summary, the few studies performed using theMycodot test indicate a high specificity (in contrast with serological tests for other, protein, antigens), buta low sensitivity. Although people with active TB have been shown to produce antibodies with a lowaffinity to the surface of live M. tuberculosis and with a low ratio of IgG/IgM [10], there is evidencesuggesting that specific antibodies might prevent M. tuberculosis dissemination.

Ongoing efforts to circumvent the technical limitations and render LAM detection inurine a useful test for TB patientsIntensive efforts are presently being made by several research teams to develop a true POC test for TBdiagnosis and patient management based on the simple detection and/or quantification of LAM in urine,with promising progress.

Increasing the diagnostic sensitivity of urinary LAM testsSince the concentration of LAM in urine from HIV-negative adult TB patients is very low, in most cases,the analytical sensitivity of the method used is absolutely crucial. We, and others, have found that the trueconcentration of LAM in urine is in the range of 15 pg·mL−1 to several hundred ng·mL−1 [60, 96–98].Based on our findings that urine concentration renders LAM detectable for most “ordinary” TB patients [47],we developed a simple concentration method. This was based on very efficient monoclonal anti-LAMantibodies in combination with a concentration step using gold coated nanoparticles [97]. With this method,we reached a sensitivity of 82% in HIV-negative patients with active TB. However, some of the componentsof the test were not stable enough, resulting in a shelf life of the test of about 3 months. Second, as shownnext, when the analytical sensitivity increased, the “matrix” effect of the urine became more prominent(i.e. the LAM signal was masked apparently by as yet unidentified components in the urine).

Other approaches to increase the sensitivity and/or the simplicity of the test have been made [99], namely:1) immunoassays exploring single-molecule fluorescence-linked immunosorbent assay [100];2) waveguide-based optical biosensors [101]; or 3) electrochemical detection on a disposable chip [102].Efforts to engineer new anti-LAM antibodies with higher affinity have for instance resulted in recombinantantibodies [103]. None of these approaches have so far reached sufficient sensitivity for clinical use. Morerecently a method applying a copper complex dye within a hydrogel nanocage that captures LAM wasdeveloped, and a sensitivity of 96% and specificity of 81% for diagnosis of pulmonary TB disease inHIV-negative adults was reported [104].

Matrix effect and how to overcome itIn experimental studies we found that urine from TB patients and healthy individuals inhibit LAMdetection at varying degrees, when purified LAM was added to different urine samples. Data shown infigure 4, using a set of 15 urine samples, clearly shows that the matrix effect varies between urine samples,LAM in the spiked urine being detectable for some individuals (see for example samples 1, 2, 9, 100 and11 in figure 4) while for others it is undetectable (see for example samples 3 and 4 in figure 4). Theinhibitory effect (“matrix” effect) varied not only between individuals, but also between samples taken atvarious time points from the same individual (data not shown).

The matrix effect is extremely important for the measurement of urine analytes. The composition of humanurine is highly variable [105] with large variations in concentration of individual components [106, 107],

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containing both organic and inorganic constituents, such as carbohydrates, amino acids, peptides, lipidsand end products of metabolism [108]. These organic entities of the urine matrix, as well as pH andelectrolytes, vary depending on normal physiological variations, health conditions, as well as differences infood consumption and medication intake. In immunoassays the matrix components may mask and/orchange the conformation of either antibodies or LAM epitopes in the assay. This masking effect may varydepending on the antibody and/or of LAM epitopes detected by distinct antibodies as well as the type ofassay used [109]. Reducing the inhibitory effects of patient urines obviously would increase the diagnosticsensitivity and accuracy of a LAM-based urine test.

The matrix effect on the LAM signal is evident also in serum [110]. The matrix effect in the serum hasbeen considered to depend on the faculty of ManLAM to form complexes with antibodies [111] and otherproteins. Using spiked samples, SAKAMURI et al. [112] demonstrated that LAM forms complexes withhigh-density lipoprotein present in the serum. In the case of serum various methods of pre-treatment havebeen tried to overcome the matrix effect using different pre-treatment schemes designed to disruptcomplexing with proteins and other components in serum and shown to improve detection [110, 113].Perchloric acid-based pre-treatment was shown to significantly improve the ability to detect LAM [110].

The importance of pHLow pH most often occurs in malnutrition [114] such as wasting TB disease and might reduce the abilityto detect LAM. We found that adjusting the pH to a neutral level had some positive effect on the LAMsignal. Data depicted in figure 4 reveal that adjusting the pH tends to improve LAM detection and that insome cases is sufficient to render LAM detectable (see for example patients 5, 7, 8 and 12 in figure 4),However, the differences in urinary pH do not exclusively explain the large variations in LAM recoverybetween different urine samples.

Effect of urine concentration on LAM detectionWe, and others, have postulated that the more the urine was concentrated the higher would be the LAMsignal. Indeed, in a recent study, GINA et al. [115] showed that collection of early morning urine (that ismore concentrated) improves the urinary lateral flow LAM assay sensitivity in hospitalised patients with

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FIGURE 4 Lipoarabinomannan (LAM) signal by ELISA in urine samples from children with suspected tuberculosis. Samples were collected atMachava General Hospital in Maputo, Mozambique, and stored at −80°C. The samples were transported frozen to the laboratory at KarolinskaInstitutet, Sweden. After thawing each urine was divided into four samples: 1) nonmanipulated; 2) spiked with 250 pg·mL−1 LAM; 3) pH adjustedwith 40 mM Tris final concentration; 4) pH adjusted and spiked with LAM. LAM signal was measured by ELISA with absorbance at 450 nmfollowing the protocol described previously [97]. PBS spiked with 250 pg·mL−1 LAM was used as a control. LAM was prepared in house asdescribed previously [79].

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TB and HIV-coinfection. However, we found that the higher the concentration of urinary creatinine,which is a marker of urine concentration, the lower the signal (data not shown), indicating that moreconcentrated urine contains more inhibitory factors. So, it seems that in patients with TB/HIV,co-infection and presumably higher concentrations of LAM in urine, the matrix does not mask the LAMsignal while in HIV-negative patients (presumably with low LAM concentration) the matrix effect isenforced by concentration. We thus tested the hypothesis that diluting urine would reduce the matrixeffect on the LAM signal, a standard method for avoiding the matrix effect in biological assays [107].Indeed, we found that the LAM signal is increased in diluted urine spiked with LAM (data not shown).However, dilution of samples with very low concentration of excreted LAM is ineffective since LAM isdiluted to levels below detection limits.

Effect of sample transportation and storageSince transportation and storage of urine samples is a key issue in clinical studies where testing iscentralised, we investigated the impact of various storage temperatures on the LAM signal in spiked urinesamples. We, and others [101], found that when the samples are stored at room temperature the signaldecreases rapidly, in a matter of hours. Even storage at 4°C causes a significant decrease in the LAMdetection as shown in figure 5 for periods as short as 1, 2 and 4 weeks. These findings are in agreementwith a study showing that after storage of urine for 2 h at room temperature LAM becomes undetectableusing an immunoblot method [101]. We, and others [116], also found that freeze–thawing extensivelyreduces LAM detection.

Actually, the fate of carbohydrate containing molecules may also be affected by chemical reactions in theurine. Thus, the formation of glucose ureide by the interaction of glucose with urea results in ammonia, anevent which may change the pH of urine. Urea in urine can, depending on the time of exposure,temperature and pH, disintegrate into isocyanates that may interact with amines on the antibodies, thuspotentially interfering with the binding of the antibodies with carbohydrates like LAM.

FIGURE 5 Effect of storage of urineat 4°C on lipoarabinomannan (LAM)signal detected by ELISA. Urine wasspiked with various concentrationsof LAM and stored at 4°C. LAMsignal was measured by ELISA withabsorbance at 450 nm following theprotocol described previously [97].

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0

Ab

so

rba

nce

at

45

0 n

m

Time of storage

Day 0

5 ng·mL–1 LAM

2.5 ng·mL–1 LAM

1 ng·mL–1 LAM

0.5 ng·mL–1 LAM

1 week 2 weeks 4 weeks

TABLE 2 Lipoarabinomannan as a potential biomarker for tuberculosis diagnosis

Type of test Present status To be explored

Urine antigen detection Low sensitivity, high specificityUseful in subgroup of HIV-positive

Solve matrix problemsIncrease sensitivity

Generate a true POC testSerum antigen detection Matrix problems

Immune complexesSolve matrix and immune complex

problemsCellular immune response Considered a potential alternative to

present IGRAMarker of protection (polycytotoxic

T-cells)Humoral immune response Low sensitivity, high specificity Usefulness for specific subgroups

Marker of protection

IGRA: interferon-γ release assay; POC: point-of-care.

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TUBERCULOSIS | M. CORREIA-NEVES ET AL.

Concluding remarksThere are several interesting venues for the use of LAM as a biomarker in the diagnosis of TB (table 2), inparticular the use of a urinary LAM-based POC test. To achieve this goal, a simple concentration step isneeded in combination with a method to circumvent the matrix effect of the urine. A method fordetection, or even quantification, of LAM in serum should be further explored.

If increased sensitivity of an antigen test (urine or serum) for LAM is achieved, LAM should be exploredas a predictive biomarker of the outcomes following M. tuberculosis infection, as well as a marker toevaluate the efficacy of anti-TB therapies.

Markers for host response to LAM (cellular and humoral) should be explored for a possible prognostic testto identify those at highest risk of progressing to active TB.

Conflict of interest: M. Correia-Neves has nothing to disclose. G. Fröberg has nothing to disclose. L. Korshun hasnothing to disclose. S. Viegas has nothing to disclose. P. Vaz reports receiving grants from the Ariel Foundation duringthe conduct of the study. N. Ramanlal reports receiving grants from the Ariel Foundation during the conduct of thestudy. J. Bruchfeld has nothing to disclose. B. Hamasur has nothing to disclose. P. Brennan has nothing to disclose.G. Källenius reports receiving grants from the Optimus Foundation during the conduct of the study and owns shares inTBDiadirect AB.

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